Recently, an increased effort has been directed toward the development of chips for molecular detection. In general, a molecular detection chip includes a substrate on which an array of binding sites is arranged. Each binding site (or hybridization site) has a respective molecular receptor (or probe) which binds or hybridizes with a molecule having a predetermined structure. A sample solution is applied to the molecular detection chip, and molecules in the sample bind or hybridize at one or more of the binding sites. The particular binding sites at which hybridization occurs are detected, and one or more molecular structures within the sample are subsequently deduced.
Of great interest are molecular detection chips for gene sequencing. These chips, often referred to as DNA chips, utilize an array of selective binding sites each having respective single-stranded DNA probes. A sample of single-stranded DNA fragments, referred to as target DNA, is applied to the DNA chip. The DNA fragments attach to one or more of the DNA probes by a hybridization process. By detecting which DNA probes have a DNA fragment hybridized thereto, a sequence of nucleotide bases within the DNA fragment can be determined.
Often, a sample to be tested will contain very few target molecules. Since there are few target molecules to identify, there can only be a few hybridization events which occur. Detecting a very low number of hybridization events can be extremely difficult. In effect, the signal to be detected will have very low gain and be difficult to separate from background noise. This problem has been ameliorated to some degree by the development of a process referred to as ligase analysis.
In ligase analysis, a DNA probe is immobilized on a support structure and immersed in a solution containing a segment of DNA carrying a marker, ligase and target DNA molecules. Under the proper conditions, the target DNA will hybridize with the probe and the segment of DNA carrying the marker. In the presence of ligase, the DNA probe and the segment of DNA will form a covalent bond. Since the target molecule is coupled to the DNA probe and the segment of DNA by a hydrogen bond, which is weaker than a covalent bond, the target molecule can be separated from the DNA probe and the segment of DNA without breaking the covalent bond coupling the DNA probe and the segment of DNA. This is typically accomplished by increasing the temperature of the solution. Once the target molecule is separated, the solution is again put in the proper condition for hybridization. The steps are repeated until the number of markers indicating a hybridization event is large enough to be detected over the background noise. In effect, the signal gain has been amplified by repeated hybridizations using the same target molecules.
There are many problems with ligase analysis. The proper conditions for hybridization can vary between target molecules. The time involved to process through a number of cycles is immense, as the solution must be slowly heated to a temperature sufficient to break the hydrogen bond while leaving the covalent bond intact and then allowed to cool for further hybridization. Furthermore, heat distribution becomes very important to uniformly heat and cool the solution and heat resistant ligase must be used so that viable ligase is present throughout the cycles.
It would be highly advantageous, therefore, to remedy the foregoing and other deficiencies inherent in the prior art.
Accordingly, it is an object of the present invention to provide a new and improved method and apparatus for identifying molecules.
Another object of the present invention is to provide a new and improved method for identifying molecules employing ligase analysis.
And another object of the present invention is to provide a new and improved method for ligase analysis of molecules, having greatly reduced cycle time.
Yet another object of the present invention is to provide a new and improved method for ligase analysis of molecules, without requiring heat resistant ligase.